Figure 1.
An HBV transcription map is illustrated.
All HBV (ayw) transcripts are shown in solid lines, and a major intron of HBV pgRNA is shown in a dashed line (HBV: 2447–489). PCR primers used for detection of all species of HBV RNAs are shown in blue (HBV: 1444–1702), and primers used for non-spliced pgRNA are shown in red and green (HBV: 2414–2606, HBV: 2301–2598). PCR primers used for detection of HBV core+ RNAs are shown in brown (HBV: 2279–2392). An HBV genome is known to encode a 3.5 kb precore specific RNA which is responsible for the production of precore and HBeAg proteins. This 3.5 kb precore transcript is longer than the 3.5 kb pgRNA by 25–30 nt at the 5′ terminus [45]. The primer pairs HBV 2301–2598 and 2414–2606 cannot distinguish between the precore RNA and pgRNA. However, our HBV replicon system (plasmid pCHT-9/3091) does not produce the 3.5 kb precore RNA [30]. Core+ RNAs refer to the 3.5 kb pgRNA, 3.5 kb precore RNA, and the 2.2 kb major spliced RNA. HBc probe (nt 1903–2447) in Northern blot analysis was specific for HBV core+ RNAs. HBV probe (nt 1521–3164) was prepared for Southern blot analysis of viral replication. C: HBV core, preC: HBV precore, P: polymerase, preS1, preS2, and S: HBV envelope, X: HBx. PRE: posttranscriptional regulatory element [19].
Figure 2.
HBV core protein (HBc) can physically and specifically associate with the cellular NXF1-p15 complex via the NES (nuclear export signal) motif of HBc arginine rich domain (ARD).
A) Upper panel: Full-length HBc (HBc 183) consists of a capsid assembly domain and an arginine rich domain (amino acid 147–183). Lower panel: HuH-7 cells were co-transfected with an HBV genome and a p15-Flag expression vector. Full-length HBc 183 protein can physically and specifically associate with a p15-Flag protein in a ribonuclease (RNase)-sensitive manner by coimmunoprecipitation assay (co-IP) and Western blot analysis (WB). B) Full-length HBc 183 protein was shown to associate with the native exogenous NXF1 protein by the same co-IP assay as described above. C) The upper panel shows a schematic outline of a K128T-HBc ARD chimera construct. The rationale of the experimental design is as detailed in the text. The co-IP result here demonstrated that only the chimera protein K128T-HBc ARD, but not K128T, can physically associate with the p15-Flag protein. D) By the same co-IP assay, only the chimera protein K128T-HBc ARD can physically associate with the native exogenous NXF1 in an RNase sensitive manner. E) Biotin-HBc ARD synthetic polypeptide can in vitro pull down purified recombinant proteins of GST-NXF1 and GST-p15 by using streptavidin T1 beads (Materials and Methods). F) A cartoon illustrates that either NXF1 or p15 can bind directly to HBc ARD. Possibility 3 refers to a situation when NXF1 and p15 do not form heterodimer even at high concentrations. The results in Fig. 2E support for possibility 4. G) Upper panel: A schematic outline of the mapped NLS and NES of HBc ARD [26]. Lower panel: HuH-7 cells were co-transfected (CoTf) with plasmid DNAs of NXF1 and p15-Flag expression vectors, and a mutant HBV genome containing R-to-A mutations at HBc NES or NLS subdomains. This NES mutant HBc exhibited significantly decreased physical association with the p15 protein (compare lane 3 with lanes 2 and 4). Ablation of the two NLS motifs appeared to increase slightly the intensity of p15-Flag (compare lane 2 and 4), suggesting that the wild type NLS could have a moderate occlusion effect on the association between p15-Flag and their neighboring NES.
Figure 3.
Association between HBc ARD and TREX complex.
A) The association between GST-HBc ARD and a TREX component ALY was ribonuclease (RNase)-sensitive and DNase-resistant in a GST pull down assay. This assay was performed by using E. coli -expressed GST or GST-HBc ARD protein and untransfected HuH-7 cell lysates (Materials and Methods). B) Full-length HBc 183 protein in HuH-7 cells transiently transfected with an HBV genome can also co-immunoprecipitate the endogenous ALY in an RNase-sensitive manner. C) HuH-7 cells were co-transfected with an HBV genome and a pCMV-DDX39 expression vector. Full-length HBc 183 protein cannot associate with another TREX component BAT1/DDX39 in the co-IP assay. D) A cartoon summarizes the putative associations among HBc, RNA, ALY, TREX components, and others (other known or unknown cellular factors). Such associations are postulated to be involved in nuclear RNA processing and export. NPC: nuclear pore complex.
Figure 4.
Perturbation of the TREX complex and NXF1-p15 nuclear export machinery by siRNA treatments can result in accumulation of HBc protein in the nucleus by IFA.
A) An HBV genome and siRNAs were co-transfected into HuH-7 cells. Transfected culture was fixed 40 hrs post-transfection before IFA. A negative control by siNonTarget treatment exhibited a cytoplasmic predominant pattern (C>N) of HBc (upper left). In contrast, siRNA treatments against NXF1 and p15 (upper right), ALY and UIF (lower left), as well as BAT1 and DDX39 (lower right), induced nuclear accumulation of HBc protein. B) Three different subcellular distribution patterns of HBc are cartoon illustrated. N>C: nucleus predominant pattern; C>N: cytoplasm predominant pattern; N+C: present in both nucleus and cytoplasm, ratio N>C/C>N: tendency of nuclear accumulation. C) Quantitative results of the IFA assay in A) were summarized in the Table. Approximately 100–150 HBc-positive cells were scored in each transfection experiment. The data shown here represent an average from at least three independent transfections.
Figure 5.
Physical association between HBV pgRNA, TREX and NXF1-p15 complex was revealed by the RNA-immunoprecipitation assay (RNA-IP).
A) The NXF1-p15 complex can associate with HBV specific RNAs by the RNA-IP assay. HuH-7 cells were co-transfected with plasmid DNAs of an HBV genome, NXF1-Flag, and p15-Flag. HBV RNAs were extracted from immunoprecipitates using anti-Flag antibody, followed by RT-PCR analysis. PCR primers 1444 and 1702 can detect all species of HBV RNAs (Fig. 1). The most abundant pre-miR-122 in HuH-7 cells was used as a negative control RNA for its lack of physical association with NXF1-p15 complex. Anti-IgG antibody (α-IgG Ab) was included as a control for the specificity of immunoprecipitation. RT: reverse transcriptase. B–D) By the RNA-IP assay, HBV 3.5 kb non-spliced pgRNA was shown to be associated with several known protein components of the NXF1-p15 mediated RNA export machinery: B) NXF1, C) ALY, and D) BAT1/DDX39. PCR primers 2301 and 2598 can detect only the 3.5 kb non-spliced pgRNA (Fig. 1).
Figure 6.
Both NXF1 and p15 can contribute to the efficient nuclear export of HBV pgRNA.
A) Upper panel: A cartoon illustrates an HBV pgRNA reporter plasmid containing a Renilla luciferase gene inserted into a major intron of HBV pgRNA [25]. Lower panel: HuH-7 cells were co-transfected with an HBV pgRNA reporter plasmid and siRNAs. Reduction of the luciferase reporter activity was observed by treatment with siRNAs specific for NXF1-p15, but not with siRNAs specific for TREX components. To control for the indirect effect of siRNA knockdown (Material and Methods), Renilla luciferase activity was normalized with a co-transfected internal reference plasmid (firefly luciferase reporter). The relative Renilla luciferase activity of mock transfection was presented as 1. The graph here represents an average from at least three independent experiments. B) Upper panel: The knockdown efficacies of siRNAs specific for p15 and UIF were measured by RT-qPCR analysis, respectively. The copy number of RNA from siNonTarget treatment was presented as 1. Lower panel: Similarly, the efficacies of siRNAs specific for BAT1/DDX39 (lane 2, left panel) and ALY (lane 2, right panel) were measured by Western blot analysis, respectively. Expression vectors pCMV-BAT1, pCMV-DDX39 (lane 3, left panel), and pCMV-ALY (lane 3, right panel), were shown to produce their respective protein products by Western blot analysis. C) Only the siRNAs specific for NXF1 and p15 reduced the cytoplasmic core+ RNA levels by Northern blot (NB) analysis using an HBc specific probe (Fig. 1). Only weak signals of pgRNA were detected in the nuclear fraction. The faint signals of 45S, 32S and 20S ribosomal RNA precursors in the nuclear fraction can be seen after longer exposure [17]. D) The knockdown efficacy of siRNAs specific for CRM-1 was measured by RT-qPCR (left panel) and Western blot analyses (right panel). The copy number of RNA from siNonTarget treatment was shown as 1. *Transfected cells in the Western blot experiment were enriched by puromycin selection. The relative distribution of pgRNA (E) and HBV core+ RNAs (F) between the nuclear and cytoplasmic compartments was measured by RT-qPCR analysis. HBV RNAs were extracted from HuH-7 cells, which were co-transfected with an HBV genome and siRNAs specific for NXF1-p15, ALY and UIF, BAT1 and DDX39. Only the siRNA specific for NXF1 and p15 exhibited a higher N/C ratio of pgRNA, while no apparent effect was observed by siRNA specific for the TREX complex. The snRNA U1 was included as an internal control to normalize the N/C ratio. N/C: relative RNA levels between nucleus (N) and cytoplasm (C). The N/C ratio of HBV pgRNA or core+ RNAs from siNonTarget treatment was shown as 1. The graph represents an average from at least three independent experiments. G) Treatment with siRNA specific for CRM-1 resulted in no effect on the N/C ratio of HBV pgRNA. The snRNA U1, known to be exported by CRM-1 [49], was used as a positive control here. GAPDH was used as an internal control to normalize the N/C ratio. The N/C ratio of HBV pgRNA from siNonTarget treatment was shown as 1. The data here represent an average from at least three independent experiments.
Figure 7.
HBV core protein (HBc) exhibited no significant effect on nuclear export of the 3.5 kb pgRNA.
A) HuH-7 cells were transfected with various plasmids of HBV genomes (based on plasmid pCHT-9/3091), containing wild type (WT) HBc, mutant ΔHBc (a core-deficient HBV genome), and a combination of ΔHBc and an expression vector of full-length HBc 183, respectively. HBV RNAs were extracted from nuclear and cytoplasmic compartments according to the Vendor’s protocol (Materials and Methods). Plasmid ΔHBc resulted in no apparent effect on the relative distribution of HBV pgRNA levels between nucleus and cytoplasm (N/C) by RT-qPCR analysis. The N/C ratio of HBV pgRNA from a WT HBV genome (plasmid pCHT-9/3091) was shown as 1. HBc protein expression was monitored by Western blot analysis (right panel). The graph represents an average from at least three independent experiments. B) Northern blot analysis revealed no significant reduction of the 3.5 kb pgRNA, in the cytoplasm of HuH-7 cells transfected with either a ΔHBc mutant or a combination of plasmids ΔHBc and HBc 183. Ribosomal RNA was included as an internal control for sample loading.
Figure 8.
Interference with the NXF1-p15 complex reduced HBV DNA synthesis.
HuH-7 cells were transiently co-transfected with an HBV genome (plasmid pCHT-9/3091) and siRNAs, followed by Southern blot analysis using HBV probe (nt 1521–3164) (Fig. 1). Upon treatment with siRNAs specific for p15 or NXF1-p15 complex, HBV replication was significantly inhibited. In contrast, no apparent effect on HBV replication was noted by treatment with various siRNAs specific for TREX components and CRM-1. RC: relaxed circle DNA, and SS: single-strand DNA.
Figure 9.
A graphic summary of the nuclear export of HBV pgRNA and HBc.
A) A cartoon illustrates a putative nuclear export pathway of HBc protein. HBV DNA genome can exist episomally as a covalently closed circular form (ccc DNA) in the nucleus, which can serve as a template for pol-II mediated RNA transcription [3]. Pre-mRNA processing and nuclear export are known to be tightly coupled by the TREX complex, which consists of ALY, BAT1, and others (other known or unknown cellular factors) [65]. HBc protein can associate with ALY, but not BAT1. Dotted line indicates that the exact sequence or molecular mechanism remains unclear. HBc protein can also associate with NXF1 and p15. It is speculated here that the NXF1-p15 export machinery can recognize a stable RNP complex between HBc protein and RNA of either viral or cellular origins. B) Nuclear export of the 3.5 kb non-spliced pgRNA is dependent on the NXF1-p15 machinery. The exact molecular mechanism of pgRNA export remains to be further investigated in the future.